High incidence of HPV infection in minors with oral squamous cell carcinoma

Background Oral squamous cell carcinoma in minors is considered to be a distinct entity from OSCC in older patients, with an uncertain etiology. Human papillomavirus (HPV) infection may trigger the initiation and promote the progression of OSCC, but these roles have not been firmly established.We aimed to explore the correlation between HPV infection and the development of oral squamous cell carcinoma in minors and know the characteristics of OSCC in young patients more thoroughly. Method From January 2013 to December 2022,6 cases of OSCC aged < 15 years were selected from the Department of Oral Pathology, Peking University School of Stomatology, Beijing, China. All cases underwent testing for high-risk HPV mRNA infection using the RNA scope technique, and immunohistochemical staining was performed to investigate the expression of p16, pan-cytokeratin (CK), CK5/6, CK7, CK8/18, epidermal growth factor receptor (EGFR), p53, and Ki-67. Furthermore, we reviewed the literature on OSCC in patients aged < 21 years. Conclusions Minors OSCC is associated with HPV infection, and that p16 can serve as an immunohistochemical marker of HPV positivity.


Introduction
Oral squamous cell carcinoma (OSCC) is a malignant tumor of epithelial origin typically found in the oral cavity that displays varying levels of squamous differentiation, as evidenced by the formation of keratinized beads and intercellular bridges.This type of cancer is most common in middle-aged and elderly individuals, less prevalent in those aged ≤ 40 years, and exceedingly rare in those aged < 15 years [1][2][3].OSCC is generally linked to tobacco and alcohol consumption, and these risk factors are not commonly present in minors.Thus, OSCC in minors is considered to be a distinct entity from OSCC in older patients, with an uncertain etiology.Moreover, human papillomavirus (HPV) infection may trigger the initiation and promote the progression of OSCC, but these roles have not been firmly established.
The treatment of OSCC typically involves a comprehensive approach, with surgical removal followed by chemotherapy and radiation therapy [4].Some studies have revealed that the prognosis of OSCC is worse for minors than for older patients, whereas others suggest that survival rates in these groups are similar.Given the rarity of adolescent OSCC, most reports describe small numbers of cases.To explore the characteristics of OSCC in young patients more thoroughly, we examined the clinical and pathological features, treatment, and prognosis of six cases diagnosed at our hospital in patients aged ≤ 15 years.All cases underwent testing for high-risk HPV mRNA infection using the RNA scope technique, and immunohistochemical staining was performed to investigate the expression of p16, pan-cytokeratin (CK), CK5/6, CK7, CK8/18, epidermal growth factor receptor (EGFR), p53, and Ki-67.Furthermore, we reviewed the literature on OSCC in patients aged < 21 years.Three cases displayed concurrent positive expression of HPV16/18 and p16.Our analysis of the clinicopathological and HPV mRNA data suggests that adolescent OSCC is associated with HPV infection, and that p16 can serve as an immunohistochemical marker of HPV positivity.

Patients
We reviewed OSCC cases in patients aged < 15 years that were diagnosed between January 2013 and December 2022 at the Department of Oral Pathology, Peking University School of Stomatology, Beijing, China.A senior pathologist validated the clinical and pathological data for these cases following surgical resection.Data on patient age and sex, tumor location, clinical manifestations, treatment, and prognosis were extracted from medical records.Of the six cases examined, one was a recurrence and five were new lesions.

Interpretation of immunohistochemical results
The staining sites of pan-CK, CK5/6, CK7, CK8/18 and EGFR were in cytoplasm [5].If more than 80% of tumor nuclei show strong positive expression or no expression at all, it is indicative of a p53 mutant; conversely, if the tumor cells show positive expression in scattered nuclei or with varying levels of intensity, it is a p53 wild type [6].Any intensity of nuclear staining indicates a Ki67 positive cell [7].In the interpretation of the p16 immunohistochemistry results, tumors exhibiting strong, diffuse staining of the nuclei and cytoplasm of > 70% of cells were classified as positive, and all other tumors were classified as negative [8].
Two oral pathology experts independently assessed all FISH and immunohistochemical results.

Clinical and radiological findings
The study sample comprised four females and two males aged 3-14 (mean, 9.8; median, 11) years.Five cases presented as primary lesions and one presented as a recurrence.Two lesions were located in the upper and lower gingiva, respectively, and the remaining four lesions were located in the mandible, maxilla, left submandibular region, and left tongue, respectively.One patient had a history of aplastic anemia, bone marrow transplantation, and previous HPV infection.The other patients had no systemic disease or family history of malignancy.
Four cases presented primarily as lumps and two cases presented as ulcers (Fig. 1A, B).Five patients underwent spiral computed tomography examinations; one patient did not undergo imaging examination at our hospital.The scans revealed bone destruction in two cases (Fig. 1C,  D), multiple abnormal lymph nodes in the neck in two cases, a malignant tumor in the left tongue and floor of the mouth in one case, and decreased bone density with visible marginal incisions in one case.Five patients had received initial medical care at local facilities, including initial surgical procedures in four cases.Biopsy results indicated that the lesions were squamous cell carcinoma in two of those cases and squamous epithelial tumor-like hyperplasia in the other two cases.
Postoperatively, two patients reported pain and lack of wound healing.One patient developed nonlocal recurrence 2 months after surgery.All patients treated at our hospital underwent surgical procedures (i.e., osteotomy, extended resection).One patient developed recurrence 1 year after the initial surgery, but not after a second surgery.One patient was lost to follow-up.The remaining patients showed no evidence of recurrence.The followup period ranged from 3 months to 5 years.Detailed clinical information is presented in Tables 1 and 2.

Histopathological findings
The cancer tissues consisted of differently sized nests with keratinized pearls congregating at the nest centers.The squamous epithelium of the lesions extended irregularly, breaching the basement membrane and infiltrating the subcutaneous connective tissue.Tumor cells varied in size and displayed eosinophilic cytoplasm with a range of squamous differentiation.Abnormal mitotic figures were frequently observed.Squamous cell carcinoma can be categorized as grade I (characterized by keratinization, few mitotic figures, and minimal cell pleomorphism), grade II (infrequent keratinization with the presence of abnormal mitotic figures), and grade III (scarcity of keratinization and abundance of abnormal mitotic figures).Four of our cases were grade I (Fig. 2A) and two were grade II-III (Fig. 2D).One case presented with lymph node metastasis and another invaded the gland, nerve, and muscle tissue.All cases were negative for extranodal extension (ENE) and positive for pan-CK, CK5/6, and EGFR.Depths of invasion were 8.9-20 mm.Three cases were positive for p16 (Fig. 2B,  E), whereas few basal layer cells and cancer nest tissues expressed p53. Ki-67 proliferation indices were 10-30%, and CK8/18 (1/6, 16.7%) was detected.All patients were negative for CK7.HPV mRNA detection with FISH, considered to be definitive for the detection of HPV infection in patients with OSCC, was conducted in all six cases and yielded positive results in three cases, consistent with the p16 expression pattern (Fig. 2C').

Literature review
Table 3 summarizes the clinical characteristics of 181 patients aged < 21 years with OSCC, extracted from the related literature.Two cases were documented in newborns.Of the patients whose ages were reported, 99 were aged ≥ 15 years and 68 were aged < 15 years.The majority (55.3%) of patients were male.The tongue was the most common site of onset, observed in 55.8% of cases, followed by the gingiva (10.5% of cases).Our sample comprised four females and two males with lesions in the tongue, gingiva, maxilla, mandible, and submandibular area.One patient was believed to have a metastasis of squamous cell carcinoma of the left mandible.

Discussion
OSCC is the predominant malignant tumor in the oral cavity, constituting almost 90% of all oral malignancies [17].Its incidence is highest among individuals aged 60-80 years, with a slightly higher prevalence in     males [87].The occurrence rate is significantly lower in individuals aged ≤ 40 years and even lower in those aged ≤ 15 years.In an emerging trend, however, OSCC is being diagnosed at younger ages.In 1936, Frank et al. [9] reported a case of tongue squamous cell carcinoma in an infant, the youngest patient with this malignancy known to date, who underwent radiation therapy.In 2011, Moubayed et al. [73] documented a case of squamous cell carcinoma in a newborn's lips.Okuyama et al. [88] analyzed the clinical and pathological characteristics of OSCC in 107 patients aged 15-39 years and 420 patients aged ≥ 70 years; they found that the tongue was most commonly affected in both groups and the floor of the mouth was also prone to lesions, with no significant sex difference.Smoking and alcohol consumption are recognized risk factors for OSCC development in adults [52,73,89], whereas these risk factors are infrequently present in minors.No patient in our sample had a history of smoking, drinking, or betel nut chewing.Several studies have suggested that squamous cell carcinoma development is more likely in children with conditions such as Fanconi anemia, xeroderma pigmentosum, and keratitisichthyosis-deafness syndrome [60,80].Individuals with xeroderma pigmentosum face a 3000-fold increased risk of developing tongue cancer compared with the general population [78].Harper et al. [19] reported a case of severe progressive xeroderma pigmentosum in an 18-year-old male who died of verrucous carcinoma of the tongue.Yagi [20] also documented a case of xeroderma pigmentosum that led to squamous cell carcinoma of the tongue in a man whose brother had the same skin condition.Research indicates that bone marrow transplantation increases the risk of solid tumor development [55].Socie et al. [34] reported that 4 of 147 patients with anemia (107 with aplastic and 40 with Fanconi anemia) who underwent bone marrow transplantation between 1980 and 1989 had developed malignant solid tumors after 64 months of follow-up.Masserot et al. [63] reported that 13 patients with Fanconi anemia developed head and neck squamous cell carcinoma after stem cell transplantation.Notably, one patient in our study with aplastic anemia who underwent immunosuppressive therapy and bone marrow transplantation developed a tongue ulcer, later diagnosed as squamous cell carcinoma, 3 years thereafter.
In recent years, HPV has been associated with squamous cell carcinoma of the head and neck, especially oropharyngeal squamous cell carcinoma.High-risk HPV cancers are driven by major HPV oncogenes, E6 and E7, which promote uncontrolled cell growth and genomic instability by down-regulating tumour suppressor genes, p53 and retinoblastoma (Rb), respectively [90].The HPV status of oropharyngeal squamous cell carcinoma lesions is considered to be an independent prognostic factor, with positivity associated with a higher 5-year survival rate and better prognosis [91][92][93].HPV has also been implicated in other types of head and neck cancer, including laryngeal squamous cell carcinoma and oral squamous cell carcinoma.Research indicated that the incidence of HPV positive OSCC had increased significantly from 1973 to 2004, particularly among white and at younger ages, the prevalence is higher among men than among women [94].In a study involving 47 OSCC patients under the age of 40, it was discovered that 15 of them were HPV positive.These patients exhibited a 5-year survival rate of 52% and a 10-year survival rate of 27.7%, which was greater than that of HPV negative patients [95].In this study, we conducted immunohistochemical detection of p16 protein and FISH detection of HPV mRNA in all cases.Three of the six patients tested positive for both p16 and HPV mRNA, and one had a history of HPV infection, and two of them received postoperative radiotherapy and chemotherapy.To date, recurrence has not developed in these three patients.Some researchers believe that the presence of HPV in OSCC had been related to improved prognosis, especially when chemo or radiotherapy is used,probably because of the absence of field cancerization or enhanced chemoradiation sensitivity [95].Nonetheless, the association between HPV infection and the prognosis of OSCC in minors remains unclear.
The diagnosis of OSCC in minors presents a significant challenge for clinicians and pathologists, primarily because the signs and symptoms of OSCC in minors often mimic those of other conditions.In addition, lesions may be detected not in the precancerous stage, but only after significant progression.Clinically, gingival inflammation in adolescent patients is frequently mistaken for an inflammatory disorder, and lesions on the tongue are often misinterpreted as ulcers or injuries.Moreover, persistent lesions or ulcers in the mouth are frequently overlooked, leading to misdiagnosis or missed diagnoses.Morris et al. [72] reported a 6-month delay of biopsy by dentists and oral surgeons in 1 of 10 tongue squamous cell carcinoma cases in individuals aged 15-20 years, resulting in the patient's presentation with a T3 tumor.Such delays can result in severe complications, such as carcinoma, and delayed treatment.Thus, the early biopsy of suspect lesions is crucial, especially in patients with histories of immunosuppression, previous tumors, radiotherapy, and/or genetic predisposition to malignancies.In cases of uncertainty, consultation with experienced pathologists and, when necessary, the prompt performance of more extensive repeat biopsy are recommended to avoid unnecessary diagnostic delays [58].Two patients in our sample presented to the hospital with postoperative wound healing failure and two presented with unhealed ulcers.Postoperative pathological examination led to the diagnosis of squamous cell carcinoma in all of these cases.Hence, the early biopsy of such lesions is vital for timely diagnosis and treatment [60].
The diagnosis of highly and moderately differentiated squamous cell carcinomas in minors can be challenging, and these lesions can be mistaken for pseudoepitheliomatous hyperplasia.This non-cancerous epithelial cell growth, which can occur due to inflammation or the presence of a tumor, is characterized by irregular epithelial bands extending into the underlying connective tissue.It may also present keratinized beads that appear to infiltrate the region.On microscopic examination, it may resemble highly differentiated squamous cell carcinoma due to the separation of the proliferative epithelium from the surface epithelium.However, cell heterogeneity is less evident and the nuclear-to-cytoplasmic ratio is not as high [46,66].In addition, squamous cell carcinoma of the jaw must be differentiated from keratinizing ameloblastoma, which can also exhibit significant keratinization on microscopic examination but lacks cell heterogeneity and mitosis.p53 and Ki-67 immunohistochemical staining can aid the diagnosis.Whereas p53 expression is reduced in keratinizing ameloblastoma, p53 immunoreactivity is high in OSCC.Furthermore, the Ki-67 proliferation index is higher for OSCC than for keratinizing ameloblastoma.The majority of ameloblastomas have been demonstrated to have BRAF V600E mutations [96].
OSCC is characterized by robust infiltration and the propensity to metastasize, and is often diagnosed at an advanced stage in middle-aged and elderly patients.The primary treatment for adult OSCC comprises a comprehensive approach including extensive tumor resection, selective neck lymph node dissection based on the presence of lymph node metastasis during surgery, and postoperative chemotherapy and radiotherapy [43,47,89,97].The use of radiotherapy to treat minors, who are in a critical stage of development, could negatively affect the soft and hard tissues in the maxillofacial region, potentially leading to facial asymmetry and even secondary malignant tumors.Consequently, comprehensive surgical resection is the preferred treatment for adolescent OSCC.Certain research suggests that postoperative radiotherapy significantly reduces local recurrence rates, prompting some experts to recommend its consideration along with proactive surgical treatment for younger patients [47].All patients in our sample who underwent radical neck dissection to remove the primary tumor and local lymph nodes were diagnosed with OSCC.The prognostic value of factors such as regional lymph node metastasis, infiltration depth, and neural invasion has been evaluated [98].Among our cases, one patient had lymph node metastasis with no ENE.This patient received postoperative adjuvant chemotherapy and radiotherapy, but developed recurrence 1year postoperatively; no recurrence has been noted since the second surgery.Throughout the follow-up period, patients underwent regular physical examinations and imaging studies to monitor recurrence.No evidence of recurrence was observed.
This study is constrained by the limited number of cases analyzed.For a better understanding of OSCC occurrence in minors, the collection of more samples and in-depth analysis of associated factors in future research are crucial.
In conclusion, these findings have found that there is a high prevalence of HPV infection among minors suffering from oral squamous cell carcinoma.Early biopsy, p16 immunohistochemical staining, and HPV mRNA detection are vital for the effective treatment and prognosis determination of OSCC.

Fig. 1
Fig. 1 Clinical characteristics and radiological features of OSCC.A and B Clinical photograph of left mandibular gingival lesion at presentation shows cauliflower-shaped lesions on the buccal and lingual side of deciduous mandibular left canine tooth (blue arrows indicate).C and D Cone Beam Computed Tomography (CBCT) showed that resorption and destruction of the bone around the roots of teeth 72 to 75 can be seen, with unclear borders and bone discontinuity at the perimeters (red arrows indicate).The buccal lingual cortex is discontinuous and visible periosteal reactions are present.Additionally, the roots of 73 and 74 teeth are floating and no external absorption is visible (red arrows indicate)

Fig. 2
Fig. 2 Representative images of histological characteristics, immunohistochemical staining and FISH.A and D Excised tissue stained with hematoxylin and eosin showing the histologic appearance of well-differentiated and middle differentiated squamous cell carcinoma.B and E Positive immunostaining for p16 in OSCC.C and F Examples of HPV mRNA detection with FISH showing yielded positive results in OSCC.(C' and F' are higher-magnification views of boxed areas) Scar Bar: 500 μm (A, B, D and E) 0.100 μm (C, F), 25 μm (C' , F')

Table 1
Clinical characteristics of OSCC in adolescentsF female, M male, y year, mo month, ER extended resection, CLND cervical lymph node dissection, LN lymph node metastasis, R recurrence, NR no recurrence, NA not available, / loss to follow-up

Table 2
Pathological and molecular findings of oral squamous cell carcinoma in adolescents

Table 3
Summary of reported oral squamous cell carcinoma cases in patients aged < 21 years

Table 3
(continued) F female, M male, y year, R right, L left, FOM floor of mouth, NS not specific